PLSCR1 is not phosphorylated by PKCδ in vitro and is not phosphorylated in apoptotic Jurkat cells. (A) PLSCR1 was incubated with recombinant PKCδ in an in vitro phosphorylation assay as described in “Materials and methods.” Proteins were resolved by SDS-PAGE and visualized by autoradiography (top panel), or immunoblotting for PLSCR1 with monoclonal antibody 4D2 (bottom panel). Histone served as positive control as a substrate for PKCδ (top, right). Note also autophosphorylation of PKCδ. (B) Jurkat T cells were incubated in medium containing [γ-³²P] ATP for 2 hours at 37°C in absence (lane 1) or presence (lanes 2-3) of anti-Fas antibody to induce apoptosis. Samples were immunoprecipitated with anti-PLSCR1 antibody 4D2 (lanes 1,3) or normal mouse IgG (lane 2). Immunoprecipitates and an aliquot of cell lysate were separated by SDS-PAGE, transferred to PVDF membranes, and either immunoblotted for PLSCR1 (left top) or visualized by autoradiography (left bottom). Extent of apoptosis was assessed by quantifying PtdSer exposure on Jurkat cells following incubation for 2 hours in presence (bold line) or absence (thin line) of anti-Fas, as measured by the binding of factor Va light chain (right panel). Histogram of mean fluorescence is shown. See “Materials and methods” for details.